Cancer cells and normal cells differ in the way they respond to DNA damage, and much of this difference is dependent on the functional integrity of the tumor suppressor p53. Recent studies have revealed that p53 has an important function in controlling the frequency of homologous recombination, in addition to its role as a transcriptional activator. In the previous funding period, we demonstrated that p53 suppressed homologous recombination, which was also independent of transcriptional activation or repression. We determined that p53 was not acting directly via the Rad51 recombinase, but through the Replication Protein A (RPA). Mutants of p53, that fail to bind RPA, retained the ability to activate apoptosis and the G1/S cell cycle checkpoint, but failed to suppress homologous recombination. We now propose to investigate this genetic dissociation of function more extensively. The link between p53 and tumorigenesis will be investigated by generating a knock-in mouse, with an equivalent p53 mutation, which will lose the ability to suppress homologous recombination but will retain the ability to activate apoptosis and cell cycle checkpoints. The impact of this cellular phenotype on the predisposition of the mouse to tumor development will be observed. If these mice develop tumors, then the control of homologous recombination would be shown to be an important step in preventing tumors, not just the ability of p53 to remove damaged cells from the replicating pool. The molecular mechanisms involved in the control of RPA by p53 will be further characterized. Phosphorylation of RPA appears to be an important step in the response to DNA damage, especially in the S-phase of the cell cycle. This phosphorylation releases p53 from RPA to allow transactivation of p53 dependent genes. The phosphorylation of RPA is under the control of a number of damage-responsive protein kinases, and the specific roles of these kinases will be determined in different cellular contexts, including phases of the cell cycle. The hypothesis is that p53 suppresses homologous recombination by its association with RPA in S-phase and there by mediates replication fidelity. Overall, we expect these studies to confirm the dual model of p53 function: a basal level of p53 controls RPA from triggering unwanted recombination events in S-phase, and that after DNA damage, p53 is released from PRA and can activate a cascade of stress responses.